Abstract

We have used paired-end sequencing of yeast nucleosomal DNA to obtain accurate genomic maps of nucleosome positions and occupancies in control cells and cells treated with 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. In control cells, 3AT-inducible genes exhibit a series of distinct nucleosome occupancy peaks. However, the underlying position data reveal that each nucleosome peak actually consists of a cluster of mutually exclusive overlapping positions, usually including a dominant position. Thus, each nucleosome occupies one of several possible positions and consequently, different cells have distinct local chromatin structures. Induction results in a major disruption of nucleosome positioning, sometimes with altered spacing and a dramatic loss of occupancy over the entire gene, often extending into a neighbouring gene. Nucleosome-depleted regions are generally unaffected. Genes repressed by 3AT show the same changes, but in reverse. We propose that yeast genes exist in one of several alternative nucleosomal arrays, which are disrupted by activation. We conclude that activation results in gene-wide chromatin remodelling and that this remodelling can even extend into the chromatin of flanking genes.

Length distribution of nucleosomal DNA. Shown are data for 3AT-treated cells. (A) Digestion of yeast nuclei with MNase (30, 60, 120 and 240 Worthington units). DNA was analysed in an agarose gel stained with ethidium bromide. M, pBR322 digested with MspI. Mono-nucleosomal DNA in the samples obtained using 60, 120 and 240 U of MNase was gel purified and repaired. An aliquot was end-labelled using T4 polynucleotide kinase and analysed in (B) a native polyacrylamide gel and (C) a denaturing polyacrylamide gel; M: 50-bp ladder (NEB). The sample obtained using 120 U MNase was ligated to paired-end adaptors and amplified by PCR (D). The purified product was used for sequencing (M: pBR322 MspI). (E) Nucleosome sequence length distribution. All sequences are included, except those derived from the yeast 2-µm plasmid. The fraction of sequences of a given length is expressed as a percentage of the total. The numbers indicate peak values. The scale is single nucleotide resolution.

Nucleosome position clusters on the PHO5 promoter, TRP1 ARS1 and GAL1-GAL10. Chromatin structures of well-studied genes. Control cells: red trace; 3AT-treated cells: green trace. Grey lines indicate 250-bp intervals. (A) Nucleosome occupancy of the PHO5 promoter and upstream PBY1 gene. All sequences are included. Nucleosomes are indicated by ovals drawn to scale and numbered (). (B) Nucleosome occupancy as in A, except that only reads of 145–155 bp were included. Nucleosome positioning analysis for (C) control and (D) 3AT-treated cells. The PHO5 coding region was omitted because it is very homologous to other yeast genes (PHO3, PHO12 and DIA3) resulting in removal of some sequences because their origin is uncertain. (E) TRP1 ARS1. Occupancy profiles for control and 3AT-treated cells (all sequences included) and position analysis for control cells. Nucleosome ovals are drawn to scale and numbered (). Our strain is trp1-1, which corresponds to a nonsense mutation (Asterisk) covered by the second nucleosome peak on TRP1, which is much reduced relative to the others, because sequences containing this mutation are rejected as they do not match the wild-type sequence. (F) GAL1-GAL10. Occupancy profiles for control and 3AT-treated cells (all sequences included) and position analysis for control cells. Black boxes: Gal4-binding sites. Grey boxes: Reb1 sites involved in expression of a ncRNA beginning within GAL10 (). The nucleosome oval is drawn to scale.

Altered nucleosome position clusters on HIS3 in response to 3AT. (A) Nucleosome occupancy on HIS3 and flanking sequences. Control cells: red trace; 3AT-treated cells: green trace. All sequences are included. Nucleosomes are indicated by ovals drawn to scale. Grey lines indicate 250-bp intervals. (B) Nucleosome occupancy using only reads of 145–155 bp. Positioning analysis for (C) control cells and (D) 3AT-treated cells. D1–D5: dominant positions adopted by nucleosomes in cells lacking the Gcn4 activator (). The asterisk indicates the new position cluster formed in place of D3 and D4 in 3AT-treated cells.

Repression of MOG1 and URA1 by 3AT results in re-ordering of disrupted chromatin structure. Occupancy profiles and position cluster analysis for two genes with disrupted chromatin structure in control cells () that are repressed by 3AT () (). Control cells: red trace; 3AT-treated cells: green trace. All sequences are included. Nucleosomes are indicated by ovals drawn to scale. Grey lines indicate 250-bp intervals. (A) Nucleosome occupancy and positioning on MOG1. (B) Nucleosome occupancy and positioning on URA1.

Alternative arrays can account for position clusters. (A) A position cluster with a central dominant position and four alternative positions. Peaks indicate nucleosome dyad positions. (B) Occupancy profile and position midpoints for an array of five perfectly positioned nucleosomes. Nucleosome: 145 bp; linker: 20 bp. Smoothed with a 25-bp moving average. (C) Occupancy profile and position midpoints for five alternative arrays of five nucleosomes: a dominant array (dark grey ovals; relative occupancy = 1); two arrays shifted by 20-bp upstream and downstream of the dominant array (light grey ovals; relative occupancy = 0.5); two arrays shifted by 40-bp upstream and downstream of the dominant array (white ovals; relative occupancy = 0.2). (D) Occupancy profile and position midpoints for two arrays of equal occupancy but different spacing, beginning and ending with the same nucleosome: upper array: five nucleosomes with 20-bp linker (165-bp repeat); lower array: four nucleosomes with 75-bp linker (220-bp repeat).